Plant cells have three gene-containing compartments: the nucleus, mitochondria and plastids. There are several types of plastid including: (1) chlorophyll-containing chloroplasts; (2) yellow, orange or red carotenoid-containing chromoplasts; (3) starch-storing amyloplasts; (4) oil-containing elaioplasts; (5) proplastids (plastid precursors found in most plant cells); and (6) etioplasts (partially developed chloroplasts that form in dark-grown seedlings). Each plastid creates multiple copies of the 75-250 kilo bases plastid genome. The number of genome copies per plastid is flexible, ranging from more than 1000 in rapidly dividing cells, which generally contain few plastids, to 100 or fewer in mature cells, where plastid divisions has given rise to a large number of plastids. The plastid genome contains about 100 genes encoding ribosomal and transfer ribonucleic acids (rRNAs and tRNAs) as well as proteins involved in photosynthesis and plastid gene transcription and translation. Most plants inherit the plastids from only one parent. Angiosperms generally inherit plastids from the mother, while many gymnosperms inherit plastids from the father. Algae also inherit plastids from only one parent. This fact allows for gene manipulation in plastids as a means of controlling inadvertent gene dispersion, a concern in genetically modified plant agriculture.
The various forms of plastid (amyloplasts, chromoplasts, etc.) have desirable properties as places to conduct reactions and to accumulate proteins or products of enzymes, and they can be exploited using the embodiments provided. Some syntheses other than photosynthesis are only carried out in plastids, probably because one or another feature of the organelle's environment does not exist elsewhere in the cell. Furthermore, the cytoplasm and chloroplast contain different proteases, and a protein might survive better in one compartment than in the other. A specific type of plastid might be a good place to accumulate certain proteins or their biosynthetic products that would be harmful if they were present in large amounts in the cytoplasm or in a plastid of a different type.
Mitochondria in plants, as in other eukaryotes, play an essential role in the cell as the major producers of ATP via oxidative phosphorylation in addition, mitochondria are involved in numerous other metabolic processes including the biosynthesis of amino acids, vitamin cofactors, fatty acids, and iron-sulphur clusters. Plant mitochondria also play crucial roles in many other aspects of plant development, performance, cell death and possess an array of unique properties which allow them to interact with the specialized features of plant cell metabolism. This includes regulation of oxidative stress during infection and pathogen attack and modulating plant responses as a means of survival during stresses such as cold temperatures, drought, and nutrient limitation. Furthermore, plant mitochondria and the accumulation of mutations in plant mitochondrial genomes, may serve as the cause of plant aging.
In comparison to plastid genomes, the size of plant mitochondrial genomes is quite variable. Furthermore, plant mitochondrial genomes can be variable even within the same plant cell, a term referred to as heteroplasmy. Owing to robust recombination rates, the plant mitochondrial genome is a highly complex structure composed of small circular and large circularly permuted DNA molecules. Plant mitochondrial genomes encode for a host of proteins and RNAs involved in various plant cell processes including oxidative phosphorylation. In the course of evolution, many organisms tackled the task of introducing macromolecules into living cells. Aside from the cell-specific, usually receptor-mediated or active uptake mechanisms, the general solution that has independently emerged in many lineages relies on peptides specifically evolved to interact with, and insert into lipid bilayer membranes. Thus, bacterial colicins, human porins, and protein transduction domains (PTDs) from diverse species share the motif of a positively charged alpha-helix, frequently with an amphipathic structure, which is capable of inserting into lipid membranes, and delivering larger cargoes intracellularly. Recent research reports confirm the successful use of PTDs fused to proteins for their delivery across biological boundaries, including the blood-brain barrier, and the placenta.
Another issue of great importance in the delivery of macromolecules in organisms is the need to protect them from proteolytic, nucleolytic and immune degradation and removal while traversing extracellular spaces. An often used approach is coating DNA with proteins capable of surviving the harsh journey to the target. Viral capsid proteins have been quite successful, yet for the purpose of DNA delivery in humans they suffer from a significant drawback—immunogenicity, the capacity to evoke a strong immune reaction greatly reducing the effectiveness of gene therapy.
Thus, there is a need for improved compositions and methods for the delivery of polynucleotides to the interior of a cell.